We have characterized and cloned two carboxypeptidases present in monocytes/macrophages and endothelial cells, carboxypeptidases M (CPM) and D (CPD), that cleave C-terminal arginine (Arg) from peptides or proteins. We have also found that enzymatic release of Arg can regulate the synthesis of nitric oxide (NO). As the enzymatic release of Arg and the production of NO at sites of injury may be critical in the regulation of pulmonary vascular endothelial barrier function, and based on our preliminary findings, we will test the following hypothesis: cellular carboxypeptidases increase the availability of Arg to NO synthase and amplify NO synthase and amplify NO production, which acts as an autocrine and paracrine signal to alter pulmonary vascular endothelial permeability. In Specific Aim 1, we will elucidate the role of predominantly extracellular CPM and of intracellular CPD in regulating NO production in monocytes/macrophages and pulmonary vascular endothelial cells by: (i) determining the ability of specific carboxypeptidase substrates to increase NO production and of carboxypeptidase inhibitors to prevent NO production and (ii) investigating the regulation by carboxypeptidases of uptake of Arg into endothelial cells and monocytes/macrophages. In Specific Aim 2, we will investigate the functional consequences of carboxypeptidase-induced NO production on endothelial barrier function by: (i) measuring the effects of carboxypeptidase substrates and inhibitors on permeability of pulmonary vascular endothelial cells in vitro in the presence or absence of inflammatory mediators or co-cultured monocytes/macrophages; (ii) determining the effects of carboxypeptidase substrates and inhibitors on NO production and pulmonary edema formation in a perfused rat lung model. In Specific Aim 3, we will define the regulation of carboxypeptidase protein in pulmonary vascular endothelial cell and monocytes/macrophages in response to inflammatory mediators and identify the gene promoter regions responsible for this regulation. These studies will provide novel information on the role of carboxypeptidases in regulating endothelial- and macrophage-induced NO production and the role of NO in signaling changes in endothelial barrier function.